智能型石墨烯/光敏剂/CpG纳米光致免疫复合药物的构建及其介导的高效抗肿瘤作用研究

Multimodal nanomedicines are rapidly emerging as promising approaches to effectively treat malignant cancer in the field of nanobiotechnology. Photodynamic therapy (PDT), a well-known noninvasive modality, had been demonstrated to induce the systemic anti-tumor immunity. By taking this advantage of PDT and the professional antigen-presenting ability of dendritic cells (DCs), the project proposes a novel strategy to design a kind of smart photo- & immuno-responsive nanocomplexes (termed as GN-PA-CpG/PS) in the combination of polymer functionlized graphene nanosheets(GN),mitochondrion-targeted photosensitizer (PS) and an adjuant unmethylated cytidine-guanosine-containing oligonucleotide (CpG ODN). The carrier GN conjugated with charge-conversional polymer through a pH-sensitive linker can deliver PS and CpG ODN to mitochonodiron and endosome respectively, and thus enhance the photo-killing efficiencies of PS and eventurally the immuno activiation. Based on this hyposis, tumor antigens generated from breast cancer cells (MDA-MB-468)treated with GN-PA-CpG/PS upon light stimulation will be utilized to activate DCs ex vivo and explore the immunogenic properties (including the mature status, the cytokine section and the induced cytotoxic T lymphocyte(CTL) toxocity). Furthermore, the syngerstic in vivo immune therapy efficiencies of DC vaccination pulsed with PDT tumor antigen aganst mammary MDA-MB-468 tumor model will be evaluated with various biological, immulogical and medical techniques. The specific CD8+ CTL responses, IFN γ-producing CD8+ T cell populations, the CD4+CD25+ Treg cell populations will be detected to reveal the mechanism behind the immunotherapy of GN-PA-CpG/PS. This project will inevitably show the novel biomedical applications of graphene nanomaterials and provide valuable information for the development of PDT-generated DC vaccine with nanomedicine in cancer treatment.

发展新型的纳米药物共递体系用于恶性肿瘤的多模治疗是药物控释与肿瘤纳米生物医学领域的研究热点。本项目主要利用光动力治疗(PDT)的免疫效应和树突状细胞(DC)的专职抗原呈递能力，以pH敏感的具有电荷转移功能的聚合物-纳米石墨烯为载体，以靶向线粒体的光敏剂(PS)和免疫辅剂CpG ODN为组装单元，构建一种智能的纳米光致免疫复合药物,实现PS和CpG ODN分别在线粒体和内涵体中定点释放，从增强PDT抗肿瘤免疫响应和CpG ODN的免疫刺激效应两方面高效诱导生成肿瘤抗原，并冲击致敏DC细胞形成疫苗；在此基础上，建立髓源DC离体培养体系和乳腺癌小鼠荷鼠模型，利用生物学、免疫学和医学研究方法研究DC疫苗的特异性、显著增强的抗肿瘤免疫治疗效应和机制，评价其临床应用的可能性。本项目将拓展石墨烯纳米复合材料的生物医学新应用，同时对探索利用纳米共递平台研制DC疫苗高效治疗恶性肿瘤的新模式具有重要意义。

肿瘤免疫疗法通过激发机体免疫系统以消除肿瘤细胞，是最具前景的肿瘤第四大新技术疗法；肿瘤光学治疗不仅可以直接消融肿瘤，还可以通过多种机制激活机体的免疫响应。因而，构建用于肿瘤光学/免疫联合治疗的纳米药物共递体系，成为当前肿瘤高效治疗的新型策略之一。本项目主要基于功能化纳米石墨烯载体，利用光敏药物和免疫佐剂CpG寡核苷酸(ODN)，构建新型智能的纳米光致免疫复合物，一方面增强肿瘤光学治疗效率，另一方面通过CpG ODN进一步增强光学治疗产生的原位肿瘤抗原的免疫刺激效应，实现高效的肿瘤光学/免疫联合治疗，为恶性肿瘤的高效诊治提供了崭新的纳米制剂/诊疗平台。主要结果如下：(1)构建并研究了线粒体靶向的纳米石墨烯基载体，高效共载近红外光敏剂IR820和CpG ODN；在808 nm激光激发下，通过光动力/光热效应和免疫刺激效应协同杀伤小鼠乳腺癌EMT6，为恶性肿瘤的高效诊治提供了崭新的靶向纳米制剂；(2)通过自组装法构建了兼具磁共振成像(MRI)/荧光(FI)双模成像功能的石墨烯量子点杂化光致免疫复合物，在660 nm激光激发下对小鼠乳腺癌EMT6细胞具有显著的光热/免疫联合抗癌活性；(3)拓展设计了CD44受体靶向的透明质酸纳米光致免疫复合药物，从逆转肿瘤免疫耐受角度增强细胞毒性T细胞的存活率和杀伤率，为肿瘤治疗提供了一种靶向、安全的新型制剂；(4)拓展研究了酸敏感的具有电荷翻转功能的共载光敏剂/miR-21抑制剂的石墨烯基复合物，实现了光疗/基因联合抗肿瘤作用。项目相关研究成果发表在纳米生物医学领域重要期刊ACS Applied Materials & Interfaces (2018, 10: 6942-6955)、Acta Biomaterials (2017, 53: 631-642)、Expert Opinion on Drug Delivery (2016, 13: 155-165)、ACS Applied Materials & Interfaces (2014, 6: 21615-21623)等上,申请发明专利三项。项目构建的纳米光致免疫复合物方法简单快捷，效果优良，具有重要的转化应用潜能。本项目为功能化石墨烯的生物医学应用提供了新方向，推动了纳米药物的发展，为肿瘤的免疫联合治疗提供了新范例。